Fluorogenic probes are essential tools for real-time visualization of dynamic intracellular processes in living cells, but so far, their design has been largely dependent on trial-and-error methods. Here we propose a quantum chemical calculation-based method for rational prediction of the fluorescence properties of hydroxymethyl rhodamine (HMR)-based fluorogenic probes. Our computational analysis of the intramolecular spirocyclization reaction, which switches the fluorescence properties of HMR derivatives, reveals that consideration of the explicit water molecules is essential for accurate estimation of the free energy difference between the open (fluorescent) and closed (non-fluorescent) forms. We show that this approach can predict the open-closed equilibrium (pK values) of unknown HMR derivatives in aqueous media. We validate this pK prediction methodology by designing red and yellow fluorogenic peptidase probes that are highly activated by γ-glutamyltranspeptidase, without the need for prior synthesis of multiple candidates.